ABSTRACT The human immunoglobulin lambda variable locus (IGLV) is mapped at chromosome 22 band q11.1-q11.2. The 30 functional germline v-lambda genes sequenced untill now have been subgrouped into 10 families (Vl1 to Vl10). The number of Vl genes has been estimated at approximately 70. This locus is formed by three gene clusters (VA, VB and VC) that encompass the variable coding genes (V) responsible for the synthesis of lambda-type Ig light chains, and the Jl-Cl cluster with the joining segments and the constant genes. Recently the entire variable lambda gene locus was mapped by contig methodology and its one- megabase DNA totally sequenced. All the known functional V-lambda genes and pseudogenes were located. We screened a human genomic DNA cosmid library and isolated a clone with an insert of 37 kb (cosmid 8.3) encompassing four functional genes (IGLV7S1, IGLV1S1, IGLV1S2 and IGLV5a), a pseudogene (VlA) and a vestigial sequence (vg1) to study in detail the positions of the restriction sites surrounding the Vl genes. We generated a high resolution restriction map, locating 31 restriction sites in 37 kb of the VB cluster, a region rich in functional Vl genes. This mapping information opens the perspective for further RFLP studies and sequencing.

INTRODUCTION

In response to the presence of foreign molecules, antigens, the B cells of vertebrates produce immu- noglobulins (Ig or antibodies) that neutralize these antigens based on the principle of complementarity. Structurally, the immunoglobulins are formed by two identical heavy chains (55 kd each) and two identical light chains (25 kd each) held together by disulfide bonds assuming a Y shape. Each heavy and light chain has a constant (C) region at the carboxy end and a variable (V) region at the amino end (Padlan, 1994).

Differences in the amino acid sequences of the heavy chain C regions in the base of the Y generate five immunoglobulin classes (IgG, IgM, IgA, IgE and IgD), but there are only two types of light chains, kappa (k) and lambda (l). Binding with the antigens occurs at the complementary determining regions (CDR 1, 2 and 3) located at the level of the V regions of each heavy and light chain (Kabat et al.,1991).

The generation of diversity at the V regions is assured by three mechanisms: i) an inherited repertoire of germline V genes, ii) somatic recombination of V genes with diversity (D), junctional (J) segments and iii) somatic mutations of rearranged V-J or V-D-J genes (Tonegawa, 1983). In man, 40% of the serum antibodies are of the l type, indicating the important role played by the l chains in the antibody response (Lai et al., 1989). The human IGLV locus is mapped at chromosome 22 band q11.1-q11.2 (McBride et al., 1982; de la Chapelle et al., 1983) and, based on the Southern hybridization studies, the number of the Vl genes has been estimated at approximately 70 (Lai et al., 1989).

However, despite their importance for the humoral immune response and the above data, the molecular genetics of the l chains is less known than that of heavy (H) and kappa (k) chains. The data about genetic polymorphism and recombination and the possible role of the IGLV locus in auto-immune or lympho-proliferative diseases are still incomplete.

The detailed restriction mapping data of the cosmid 8.3 presented here represent a reference for further studies on RFLP of normal human populations and patients with auto-immune or lympho-proliferative diseases.

MATERIAL AND METHODS

Isolation and characterization of the cosmid 8.3

The human genomic cosmid library was kindly provided by Dr. L. Buluwela. It was prepared in the Lorist-6 cosmid vector ligating HindIII DNA fragments of about 50 kb from the human Colo 320 cells to the HindIII cleaved vector (Buluwela et al., 1988).

This library was screened with the IGLV1S1 gene probe (Alexandre et al., 1989) and several positive clones were found. One of these clones, named cosmid 8.3, was amplified and its DNA was prepared using Qiagen purification columns (Diagen, Hilden, Germany) and digested with different restriction endonucleases (Boehringer, Mannheim). The restriction fragments were electrophoresed in 1% agarose gel and transferred to nylon membranes (Hybond N+, Amersham) according to standard procedures (Sambrook et al., 1989). The filters were hybridized with the 32P-labeled Vl gene probes described below and washed under low stringency conditions. The probes used in the hybridizations were: the 5 IGLV1S1 insert probe, pVl1VD0.2, which is a 216-bp PvuII-HindIII fragment isolated from clone lLY67VlI (Alexandre et al., 1989) cloned in pUC-18, that contains the 5 region of the germline IGLV1S1 gene; the IGLV1S2 insert gene probe, pVl2EK0.3, which is a 300-bp HincII-KpnI fragment containing part of the IGLV2S2 gene cloned in pUC-18, and the IGLV7S1 insert gene probe, pVl7BL0.68, which is a 680-bp BamHI-BglII fragment isolated from pV3.3, a subclone of V4A (Daley et al., 1992b) and subcloned in pUC-12. The pseudogene VlA (Alexandre et al., 1989) and the gene IGLV5a (Berinstein et al., 1988) were identified by PCR as described below.

The cosmid 8.3 DNA was digested separately with EcoRI and HindIII. The fragments were separated by electrophoresis in 1% low-melting point agarose (Sea-Plaque GTG, FMC, USA) in 1x TAE buffer (40 mM Tris-acetate, pH 8.0, plus 1 mM EDTA). After electrophoresis the gel was stained with a diluted ethidium bromide solution and each DNA band trimmed from the gel under short time UV exposition. The blocks of agarose containing DNA fragments up to 8.0 kb were melted at 68°C for 10 min and placed at 37°C to prevent solidification.

The DNA fragments were ligated to pUC-18 or pT7T318U vectors cleaved with EcoRI or HindIII in an in-gel ligation reaction (1 ml of melted agarose containing the DNA fragment, 6.0 ml of deionized and autoclaved water, 1.0 unit (1.0 ml) of T4 DNA ligase (Boehringer Mannheim), 1.0 ml (20 ng) of vector and 1.0 ml of 10x ligation buffer. The mixture was left to stand at room temperature (25°C), for 18 h). The ligations were transfected to E. coli TG1 (Hanahan, 1983) and plated onto X-gal + IPTG-containing medium. The white clones harboring the correct size insert were identified by plasmid mini-preparations and digestion with the corresponding restriction enzyme (Sambrook et al., 1989). Each positive clone was named according to the origin of the DNA, the restriction enzyme and the size of the insert.

Restriction mapping

The plasmid mini-preparations of each cosmid subclone were digested with the HindIII, EcoRI, BamHI, KpnI, XhoI and SalI restriction enzymes (single and double digestions) and the fragments separated by electrophoresis in 0.8% agarose gel, 1x TBE buffer with ethidium bromide. The gels were photographed under UV illumination and the molecular weight of each fragment was determined considering the presence of the vector.

The restriction fragments from the cosmid 8.3 exceeding 8.0 kb in size were not subcloned and the restriction data were obtained by direct in-gel digestion. The restriction map of cosmid 8.3 was assembled by overlapping these fragments and the maps of each subclone.

RESULTS AND DISCUSSION

We identified several positive clones by screening the human genomic cosmid library from Colo 320 cells DNA (Buluwela et al., 1988) with a specific Vl gene probe insert (IGLV1S1 gene probe, Alexandre et al., 1989), and from those presenting the strongest hybridization signal we selected the cosmid clone named 8.3 for further analysis. The digestion of the DNA from this cosmid with EcoRI and HindIII restriction enzymes permitted us to measure an insert of approximately 37.0 kb (Figure 1).

We digested the cosmid 8.3 DNA separately with EcoRI and with HindIII in order to construct a precise map locating the restriction sites surrounding the six Vl gene sequences. The EcoRI digestion produced fifteen 8.0- to 0.3-kb fragments. The 1.8-, 1.6-, 1.2- and 0.3-kb fragments corresponded to the Lorist-6 vector. HindIII digestion produced nine 16.0- to 1.0-kb fragments, and the entire Lorist-6 vector was liberated in a 5.0-kb fragment (Figure 1). The fragments below 8.0 kb were subcloned in pUC-18 or pT7T318U vectors (except for the Lorist-6 fragments).

We constructed the complete restriction map for the cosmid 8.3 by overlapping the restriction maps of each subclone (Figure 2). We located 31 restriction sites. The six Vl gene sequences are dispersed among 22.8 kb in the 37.0-kb insert. The functional gene IGLV7S1 is 11.0 kb upstream from the IGLV1S1 gene.

The restriction map of the cosmid 8.3 overlaps with the map of phage clones lLY67Vl1 (Alexandre et al., 1989) and #4 (Daley et al., 1992a) and YAC clones 366F5, 105H2 and 400B5 (Frippiat et al., 1995), conferring a phage-cosmid-YAC contig. This confirms the position of the gene IGLV1S2 5.0 kb downstream of gene IGLV1S1. There is still a possibility that these genes evolved by duplication due to the sequence similarity (> 80%) and the positions of the restriction sites surrounding these genes. The two non-coding sequences (pseudogene VlA and vestigial sequence vg1) are located 2.0 kb and 7.0 kb, respectively, downstream of gene IGLV7S1 (Alexandre et al., 1989; Chuchana et al., 1993). Gene IGLV5a is located 4.3 kb downstream of the IGLV1S2 gene (Frippiat et al., 1995).

Cosmid 8.3 is of particular interest since it covers a region of the cluster VB, IGLV locus, which is rich in functional Vl genes and two non-coding sequences (Figure 2, see cluster B). Several haplotypes were revealed by RFLP involving polymorphic insertion/deletion of genes IGLV1S1 and IGLV7S1 (Chuchana et al., 1993) and IGLV5a (Frippiat et al., 1995). Since these genes are present in cosmid 8.3, and with the more complete restriction map for this region and the collection of subclones of this cosmid, we can now do sequencing do perform RFLP studies of the normal human Brazilian population, and in patients with auto-immune or lympho-proliferative diseases.

While this paper was under revision, Kawasaki et al. (1997) published one-megabase sequencing of IGL locus representing the longest contiguous stretch of human DNA analyzed to date.

ACKNOWLEDGMENTS

This work was carried out in the Laboratoire dImmunogénétique Moléculaire (LIGM), Institut de Génétique Moléculaire de Montpellier, France (CNRS and Université de Montpellier II), during a post-doctoral period of training of G.A.S.P. Jr who was the recipient of a fellowship from CAPES, Brasil. Research supported by the Centre National de la Recherche Scientifique, the Ministère de lEnseignement Supérieur et de la Recherche, the Université Montpellier II and Association pour la Recherche sur le Cancer (France). G.A.S.P. Jr received grants from FAPESP (No. 95/9839-3) and CNPq-FAPESP (No. 96/5842-2). Publication supported by FAPESP.